Objective To construct a folic acid deficient model in zebrafish and to observe the axial development in folic acid deficient embryos, so as to probe the mechanism by which folic acid deficiency induces abnormal development of axis. Methods We constructed the folic acid deficient zebrafish model by both using the antagonism of dihydrofolate reductase (MTX) and knocking-down dihydrofolate reductase gene. Then we observed the axial excursion of folic acid deficient embryos at 17 hpf under microscope. We labeled and observed the positions of liver, spleen and heart by using whole-mount in situ hybridization with specific antisense RNA probes. The expressions of some genes, which are down stream factors of Nodal signal pathway and important for axial development, were detected by whole-mount in situ hybridization and Real-time PCR. Results Parts of folic acid deficient embryos had axial excursion and abnormal positions of liver, spleen and heart. The expressing intensities of ntl and gsc appeared normal in folic acid deficient embryos, but the expressing spatial patterns were abnormal, which revealed the malformation of axial mesoderm. Conclusions Folic acid deficiency induced the abnormal development of axis and the malformation of axial mesoderm. Folic acid deficiency had no obviouse effect on Nodal pathway.

Objective To observe the development of hematopoietic stem cell and the apoptosis of ICM(intermediate cell mass) in folic acid deficient zebrafish embryos and investigate the mechanism by which folic acid deficiency induces abnormal hematopoiesis.Method The folic acid deficient zebrafish model was induced by both using the dihydrofolate reductase antagonism methotrexate(MTX) and knock-down dihydrofolate reductase gene.The development of embryos was observed under microscope.The blood cells were detected by O-dianisidine staining.Whole-mount in situ hybridization and real-time PCR were performed to examine the expression of FLK-1,GATA1and GATA2.Apoptosis in intermediate cell mass(ICM) was examined by TUNEL(terminal-deoxynucleotidyl transferase mediated nick end labeling) method.Results The abnormal developments of ICMs were observed both in MTX treated embryos and DHFR knock-down embryos.O-dianisidine staining revealed that folic acid deficiency resulted in the decreasing number of blood cells.In folic acid deficient embryos,the expression of FLK-1、GATA1and GATA2 was reduced and the apoptosis in ICMs was increased.Conclusion The abnormal hematopoiesis in zebrafish induced by folic acid deficiency is related with the increasing apoptosis in ICMs and decreasing expressions of FLK-1,GATA1and GATA2.

Objective: To investigate the expression of ATP-Binding Cassette Proteins including P-gp (P-glycoprotein), MRP1 (multidrug resistance associated protein 1) and BCRP (breast cancer resistance protein) in hepatocellular carcinoma and its relationship with pathological features. Methods: The expression of P-gp/MDR1 (multidrug resistance gene 1), MRP1 and BCRP in hepatocellular carcinoma was examined by RT-PCR and immunohistochemistry in 34 cases of hepatocellular carcinoma and 19 cases of paraneoplastic hepatic tissues. Results: The expression of MDR1, MRP1 and BCRP mRNA (messenger ribonucleic acid) was 1.15±0.24, 0.64±0.33, and 1.07±0.32 in hepatocellular carcinoma and 0.36±0.14, 0.19±0.06, and 0.31±0.09 in paraneoplastic hepatic tissues. The expression of MDR1, MRP1 and BCRP mRNA was 1.38±0.26, 0.73±0.35, and 1.34±0.21 in poorly differentiated hepatocellular carcinoma and 0.74±0.32, 0.30±0.11, and 0.45±0.13 in well differentiated hepatic tissues. The immunohistochemical positive substance was detected in the plasma membrane and cytoplasm. The positive rates of P-gp, MRP1 and BCRP were 82.35%, 58.82%, and 79.41% in hepatocellular carcinoma and 42.11%, 26.32%, and 36.84% in paraneoplastic hepatic tissues, respectively. The positive rates of P-gp, MRP1 and BCRP were 100.00%, 81.25%, and 100.00% in poorly differentiated hepatocellular carcinoma and 66.67%, 38.89%, and 61.11% in well differentiated hepatic tissues. The expression of three indicies in hepatocellular carcinoma was higher than that in paraneoplastic hepatic tissues (P<0.05). The expression of P-gp/MDR1, MRP1 and BCRP in poorly differentiated hepatocellular carcinoma was higher than that in well differentiated hepatic tissues (P<0.05). No correlation was found among the three indices. Conclusion: Intrinsic multidrug resistance exsists in hepatocellular carcinoma, with various mechanisms. The multidrug resistance of HCC (hepatic cell carcinoma) is related to P-gp/MDR1, MRP1 and BCRP. MRP1 and BCRP may be targets for reversing multidrug resistance.

Objective To explore the possibility that rat bone mesenchymal cells (BMSCs) can differentiate into hepatocytes under the affection of fetal liver filtrate. Methods PAS and green indigo dye were used to detect glycogen and differential level of hepatocytes, respectively. The concentration of ALT, AST, ALP in the culture supernatant were served as markers of hepaocyte function. Results Fourteen days after induced by the fetal liver filtrate, BMSCs changed their shapes into polygon, oval or round. Some of BMSCs were positive for AFP and ALB at 7 days after induction, then the number of positive cells increased, and most of BMSCs expressed AFP and ALB till 21days. The PAS reaction and indocyanine green(ICG) intaking also appeared at 7days. Enzyme in supernatant such as ALT, AST, ALP were fristly detected at 7days and peaked at 14days,then the level declined. Conclusion The fetal rat liver filtrate was able to induce BMSCs into cells with function and characteristics of hepatocytes.

BACKGROUND: There is an intimate temporal and spatial relationship between growth of primitive cardiac cells, septum transversum mesenchyme and liver development. The signal from primitive cardiac cells and septum transversum mesenchyme induces the ventral foregut endoderm cells specialize toward hepatocytes. While the septum transversum mesenchyme provides a suitable environment for forming the liver bud and promoting the growth and differentiation. However, the molecular mechanism of this induction is not yet delineated.OBJECTIVE: Using alpha-fetal protein (AFP), c-Met and cytokeratin (CK) 19 as markers of hepatic stem cells, the growth of early human embryo of 3-5 weeks and morphologic characteristic of hepatic stem cells were observed to demonstrate the characteristic and factors that affected the proliferation and differentiation of hepatic stem cell, which provided experimental evidence for basic research and clinical application of hepatic stem cells.DESIGN: An opening experiment was designed.SETTING: Department of Anatomy, Weifang Medical College.MATERIALS: The experiment was carried out at the Scientific Research Center of Chengdu Medical College between September 2004 and January 2005. Twenty cases fresh human embryos aged less than 2 months were collected with signed agreements of the pregnant women suffering from pregnancy termination with mifepristone. The samples were fixed with 40 g/L polymerisatum in 20 minutes and embedded routinely in paraffin, and then 5 μm thick series sections were continuously made. After hematoxylin-eosin staining, the embryonicage was determined under the microscope according to the length of embryos, the number of somites and the development of organs, which was referring to the Jirasek's staging standard of human embryo.METHODS: The immunohistochemical staining was conducted with SABC method on one of every ten sections, which were incubated overnight at 4 ℃ with polyclonal antibodies against hepatocyte growth factor (HGF),c-Met, insulin-like growth factor (IGF-Ⅰ), IGF-Ⅰ receptor (IGF-IR), transforming growth factor (TGFβ1), TGFβR1, TGFβR2 or monoclonal antibodies against proliferating cell nuclear antigen (PCNA), AFP and CK19.The following day, the sections were incubated for 2 hours at room temperature with biotinylated anti-mouse or anti-rabbit IgG and SABC liquid respectively, and then diaminobenzidine (DAB) was used to color them. The negative control was conducted with the phosphate buffer, then the sections were observed and photographed under light microscope.MAIN OUTCOME MEASUERS: ①the morphologic characteristic of human hepatic stem cells and immunohistochemical staining of markers②the expression of HGF, IGF-Ⅰ, TGFβ1 and their receptors on human embryonic livers of 3-5 weeks, primitive cardiac cells and septum transversum mesenchyme.RESULTS: ①The morphologic characteristic of human hepatic stem cells and immunohistochemical staining of markers: The hepatic bud formed at the end of 3rd week and migrated into the septum transversum mesenchyme to form the hepatic cords at the 4th week. The cells structuring the hepatic cords displayed the typical characteristic of immature cells. At the 5th week, the number of cells within the hepatic cords, the size of cell body,the cytoplasmic acidophilia all increased, whereas the basophilia of nuclei decreased. However the cellular forms were still homogeneous and displayed the typical characteristic of immature cells. The cells of hepatic cords were negative for PCNA response during 3rd-4th week but began to express positive at the 5th week, mainly in the nucleus and minority cellular cytoplasm showed weak positive. Most hepatic cells during 3rd-5th weeks were positive for AFP, c-Met and negative for CK19. The immunologic reaction depositors of AFP positive cells were located in the nuclei, cytoplasm and membrane of the hepatocytes, and c-Met presented mainly in the nuclei and the positive signal was weak in the cytoplasm. ②Expressions of HGF, IGF-Ⅰ, TGFβ1 and their receptors in the embryonic human liver, primitive heart and septum transversum mesenchyme: At the 4th week,the c-Met expressed only in all hepatocytes, whereas the other growth factors and their receptors were undetectable. At the 5th week, all the growth factors, except HGF, were expressed in the hepatocytes. The immunologic reaction depositors of TGFβ1, TGFβ1R1 and TGFβ1R2 were located in the cytoplasm and cell membrane. The positive response of IGF-Ⅰ and IGF-IR were present at nuclei, cytoplasm and cell membrane. At the 3rd-5th week, myocardial cells surrounding liver bud or hepatic cord and the septum transversum mesenchyme were positive for HGF, TGFβ1 and IGF-Ⅰ,with the signals were aggregated mainly in cytoplasm and minority nucei.CONCLUSION: ①It was at the end of 3rd week that part of endoderm cells in foregut ventral were specialized to hepatic stem cells. ②The undifferentiated hepatic stem cells could be drawn to develop to the liver stem cells with bi-directional differentiation potentials by using specific markers for studying human embryonic liver stem cells. According to the corresponding relation of embryonic age between human and rats, the time studying the rat hepatic stem cells could be calculated. ③HGF, IGF-Ⅰ,TGFβ1 and their receptors promoted the early development of human embryonic liver.

BACKGROUND: Vascular endothelial growth factor family and its receptor play an important role in the process of angiogenesis and neovascularization. Recently, the effect of vascular endothelial growth factor family on blood has been paid much attention. OBJECTIVE: To observe the expression of vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor C (VEGFC) and their receptors as well as angiopoietin-1 (ang-1),angiopoietin-2(ang-2) and their receptors in the blood island of yolk sac and aorta-gonadalmesonephros (AGM) region at embryonic 3 to 12 weeks, so as to investigate the effect of VEGF and angiopoietin family in the process of haematogenesis. DESIGN: Single sample observation. SETTING: Weifang Medical College, Experimental Center of Morphology and Staff Room of Anatomy. MATERIALS: This experiment was carried out at Weifang Medical College from February 2002 to August 2004. Specimens were collected from 47 pregnant women's 3-to-12-week abortive embryos. Informed consent was obtained from the pregnant women. METHODS: Specimens were performed successive sections. Two sections were drawn out every other 10 sections. Hematoxylin-eosin staining and immunohistochemical staining were performed. Polyclonal anti-vascular endothelial growth factor C, fms like protein tyrosine kinase(PTK), flt-4,ang-1, ang-2,Tie-2 antibody, monoclonal anti-vascular endothelial growth factor A and fetal liver kinase 1 (flk1) antibody were used for incubation overnight at 4 ℃ .Goat anti-mouse IgG and SABC solution were used separately for 2 hours. 3,3'2 diaminoazobenzidine (DAB) was used to develop. Serum of normal rabbit or mouse were used separately to replace primary antibody as negative control. They were observed and taken photographs under optical microscope. MAIN OUTCOME MEASURES: ①Blank expression of VEGFA and VEGFC and their receptors in the blood island of yolk sac and AGM region at embryonic 3 to 12 weeks. ②Blank expression of ang-1 and ang-2 and their receptors in the blood island of yolk sac and AGM region at embryonic 3 to 12 weeks. RESULTS: ①On the 21st to 25th day , vascular endothelial cells and hematopoietic cells in blood island of yolk sac strongly expressed VEGFA and its receptor fms like PTK and flk1, VEGFC and its receptor flk4, ang2 and its receptor Tie-2 protein, weakly expressed ang-1 protein. ②From the fourth week of development, dorsal aorta and mesonephros expressed above-mentioned various factors and their receptors . Immune positive reactants gathered in the large and round hematopoietic cells with nucleus. The cell quantity reached the peak at week 7. After week 8, the number of positive cells was significantly decreased, and almost all the blood cells were immune negative reaction at week 12. ③Gonad mainly expressed VEGFA , fms like PTK, flt-4,ang-1 ,ang-2 and Tie-2 protein at weeks 6 to 8 , but did not express VEGFC and flkl. ④ The expression of above-mentioned factors were not detected in the vascular endothelial cells in AGM region. CONCLUSION: Hematopoietic cells and endothelial cells of blood island of yolk sac of human embryo as well as dorsal aorta and mesonephros coexpresses various factors related to angiogenesis and haematogenesis. Haematogenesis of human embryos occurs at the fourth week.

BACKGROUND: Studies of biological characteristics of mesenchymal stem cells (MSCs) and regulatory factors that influenced the differentiation of MSCs have shown that the proportion of the natural differentiation from in vitro primarily cultured MSCs into hepatocytes was low, and to select a suitable inductor is important to enhance the differentiation of MSCs into hepatocytes.OBJECTIVE: To verify the feasibility of induced differentiation of rat bone marrow MSCs (BMSCs) into hepatocytes using the combination of hepatocyte growth factor (HGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF-4).DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Experimental Center, Weifang Medical College in August 2007.MATERIALS: Totally 40 Sprague-Dawley rats were supplied by the Experimental Animal Center, Weifang Medical College.METHODS: Rat BMSCs were incubated by adherent method. BMSCs at passage 3 were assigned to 2 groups. BMSCs in the blank control group were treated with L-DMEM containing 10% fetal bovine serum. BMSCs in the combination group were treated with 10 μg/L FGF, 8 μg/L HGF and 8 μg/L EGF following above-mentioned procedures.MAIN OUTCOME MEASURES: Inverted microscope was used to observe the morphological changes in cells.Immunofluorescence method was used to observe the expression of alpha-fetoprotein (AFP) and albumin (ALB). PAS was employed to detect the expression of glycogen. Fox green intake experiment was conducted. Enzymology was utilized to test the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP).RESULTS: BMSCs in the combination group presented polygonal, orbicular or round shape. BMSCs in the blank control group remained spindle. BMSCs in the combination group were positive for AFP and ALB at day 14 following culture, and a few PAS-positive and fox green-positive cells were found at day 7. Positive cells became more over time. Synthesis of ALT, AST and ALP was detected at day 14, reached a peak at 21 days, and then decreased. Above-described indexes were negative in the blank control group.CONCLUSION: After induced by the FGF, HGF and EGF, BMSCs have the ability to differentiate into hepatocytes in vitro.

BACKGROUND: There are few studies conceming morphological charactedstics, space-time distribution and differentiation of hepatic stem cells during embryo liver development.OBJECTIVE: To understand the action of alpha fetoprotein, cytokeratin-19 (CK19) and c-Met in the liver through observing the expression of them.DESIGN, TIME AND SETTING: The in vitro cytological observational study was performed at the Central Laboratory of Weifang Medical College from June 2005 to December 2006.MATERIALS: A total of 40 embryo samples were obtained from 3-rnonth aborted fetus, which were supplied by Hospital Affiliated to Weifang Medical College.METHODS: Aborted embryo was collected and made into sections within 30 minutes. Fetal age was defined according to embryonic layer formation, somite number and organ development under a microscope. Sample sections with fetal age of 3-12weeks were selected. One was collected from every eleven sections and underwent immunohistochemical staining.MAIN OUTCOME MEASURES: Expression of alpha fetoprotein, CK19 and c-Met was measured in embryo hepatic stem cells aged 3-12 weeks.RESULTS: At 3-5 weeks, samples were positive for alpha fetoprotein and c-Met, which were indicated as hepatic stem ceils. At 10-12 weeks, alpha fetoprotein- and c-Met-positive cells were mainly distributed surrounding the header, which suggested that hepatic stem cells were mainly located at hepatic cord of the header. This had similar distribution as adult hepatic oval cells (adult hepatic stem cells). CK19-positive reaction was found at week 7, and mainly at hepatic cord cells, bile duct sheet cells and bile duct epithelial cells at 10-11 weeks. CKlg-positive reaction was only seen at the bile duct sheet and bile duct epithelial cells at week 12. At this time, all bile duct sheet cells and bile duct epithelial cells were positive for alpha fetoprotein, c-Met and CK19.CONCLUSION: CK19-positive reaction was not found in hepatic stem cells, but only detected in bile duct epithelial cells and progenitor calls. CK19 may be not fit for a marker of hepatic stem cells. All bile duct sheet and bile duct epithelial cells are positive for alpha fetoprotein, c-Met and CK19. It is assumed that alpha fetoprotein+/c-Met+/CK19+ may be bile duct progenitor calls.

Objective To study the putative mechanisms underlying fetal alcohol syndrome by comparative protein-profile analysis between normal and ethanol-treated zebrafish embryo with twodimensional electrophoresis (2-DE).Methods Zebrafish embryos were exposed in 400 mmol/L ethanol at dome stage for 3 hours,and then ethanol-induced abnormalities were observed.Proteomes of zebrafish embryos at early stages including zygote stage,dome stage,shield stage and 5-somite stage,were separated by 2-DE.The subtraction analysis method was applied to eliminate the interference from maternal derived proteins.The ethanol-treated embryos at 5-somite stage was analyzed by 2-DE,and the protein profile was compared with that generated from control embryos at the same stage.The data obtained from 2-DE analysis were verified by in-situ hybridization.Results 400 mmol/L ethanol treatment caused axial malformation (62%) and cyclopia (60%) in zebrafish embryos.The 2-DE analysis showed that the expression of Collagen2al (Col2a1) and TAR DNA binding protein (TDP) was decreased in 12 hours post fertilization (12 hpf) ethanol-treated embryos by 81% and 73%,respectively.The in-situ hybridization also demonstrated that the expression of Col2al in axial mesoderm was reduced by ethanol treatment at the same stage.But for 24 hpf ethanoltreated embryos,the expression of Col2al in axis recovered to a comparable level to that in control embryos,while the structure of neural tube was disrupted severely by ethanol exposure.Conclusions It is suggested that the expressions of Col2al and TDP were disrupted by ethanol during early stage,which might induce the zebrafish developmental abnormalities.The ethanol interference on early expression of Col2al is supposed to be one of the major reasons leading to later abnormalities of axis and neutral tube.

Objective To select the tag SNPs of TLR2 gene in the Chinese population with bioinformatics techniques.Methods We ascertained the assayed scope of the TLR2 gene with the aid of NCBI database and downloaded SNP genotype data of TLR2 gene in the Chinese population from Hapmap database.Then Haploview (version 4.0) was used to calculate linkage disequilibrium (LD) statistics.Haplotype blocks were constructed throughout the TLR2 gene according to the upper and lower 95% confidence bound of the D'value.Meanwhile,we selected the tag SNPs based on r~2 values and the LOD value between SNPs and picked up the representative haplotypes in accordance with the proportion of each haplotype in the haplotype blocks,respectively.Results We constructed 2 haplotype blocks within the TLR2 gene and selected 3 tag SNPs containing 3013 A/G,19216 T/C and 22215 G/T in the Chinese population.Meanwhile,we identified the representative haplotypes of which the tag SNP would be on behalf of every haplotype block.Conclusion The SNPs of 3013 A/G,19216 T/C and 22215 G/T,the most representative SNPs in the whole TLR2 gene in the Chinese population,could be selected as tag SNPs to guide their association studies between the TLR2 gene and sepsis.